Anti-TLR4 antibodies and methods of use thereof

ABSTRACT

This invention relates generally to antibodies that specifically bind Toll-like Receptor 4 (TLR-4), and to methods of using the anti-TLR4 antibodies as therapeutics and to methods of using the anti-TLR4 antibodies in methods of preventing transplant rejection and/or prolonging survival of transplanted biological material.

RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. ProvisionalPatent Application No. 61/431,191, filed Jan. 10, 2011, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to antibodies that specifically bindToll-like Receptor 4 (TLR-4), and to methods of using the anti-TLR4antibodies as therapeutics and to methods of using the anti-TLR4antibodies in methods of preventing transplant rejection and/orprolonging survival of transplanted biological material.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “424001USSeqList.txt,” which wascreated on Jan. 9, 2011 and is 41.7 KB in size, are hereby incorporatedby reference in their entirety.

BACKGROUND OF THE INVENTION

Organ and tissue transplantation is the preferred clinical approach totreat patients suffering from organ failure or complications arisingfrom diseases of specific organs and tissues. However, transplantpatients face a lifetime of immunosuppressive therapy and the risk oflosing the new organ due to rejection. Although improvements have beenmade in the transplantation process, rejection remains the most commoncomplication following transplantation and is the major source ofmorbidity and mortality. Transplant rejection occurs when the immunesystem of the recipient of a transplant attacks the transplanted organor tissue. Rejection is an adaptive immune response and is mediatedthrough both T lymphocyte-mediated and humoral immune mechanisms.

Thus, there remains a need for methods to promote organ or tissuetransplantation tolerance in patients.

SUMMARY OF THE INVENTION

The invention provides methods of inhibiting rejection of and/orprolonging survival of transplanted biological material in a subjectusing antibodies that specifically bind Toll-like receptor 4 (TLR4).

The invention provides methods of inhibiting rejection of and/orprolonging survival of transplanted biological material in a subject bycontacting the biological material to be transplanted with an antibodyor immunologically active fragment thereof that specifically binds aToll-like receptor 4 (TLR4) polypeptide to produce a transplantablecomposition, and implanting the transplantable composition at a desiredlocation in the subject.

In some embodiments, the methods also include the step of administeringto the subject who has been implanted with the biological material oneor more additional doses of an antibody or immunologically activefragment thereof that specifically binds TLR4, wherein the antibody isadministered in an amount sufficient to prevent transplant rejection orprolong survival of the transplanted biological material in the subject.The additional dose of anti-TLR4 antibody can be administered during thetransplant, after the transplant or both.

The invention provides methods of inhibiting rejection of or prolongingsurvival of transplanted biological material in a subject by contactingthe biological material to be transplanted with an antibody orimmunologically active fragment thereof that specifically binds aToll-like receptor 4 (TLR4) polypeptide to produce a transplantablecomposition, implanting the transplantable composition at a desiredlocation in the subject, and administering to the subject one or moreadditional doses of an antibody or immunologically active fragmentthereof that specifically binds TLR4, wherein the antibody isadministered in an amount sufficient to prevent transplant rejection orprolong survival of the transplanted biological material in the subject.The additional dose of anti-TLR4 antibody can be administered during thetransplant, after the transplant or both.

The invention also provides methods of treating a subject who hasreceived or will receive a transplant of biological material byadministering to the subject one or more doses of an antibody orimmunologically active fragment thereof that specifically binds aToll-like receptor 4 (TLR4) polypeptide, wherein the antibody isadministered in an amount sufficient to prevent transplant rejection orprolong survival of the transplanted biological material in the subject.

In some embodiments, the subject is a mammal. In a preferred embodiment,the subject is a human.

In some embodiments, the TLR4 polypeptide is a human TLR4 polypeptide.In some embodiments, the human TLR4 polypeptide comprises the amino acidsequence:

(SEQ ID NO: 11) 1mmsasrlagt lipamaflsc vrpeswepcv evvpnityqc melnfykipd nlpfstknld 61lsfnplrhlg sysffsfpel qvldlsrcei qtiedgayqs lshlstlilt gnpiqslalg 121afsglsslqk lvavetnlas lenfpighlk tlkelnvahn liqsfklpey fsnltnlehl 181dlssnkiqsi yctdlrvlhq mpllnlsldl slnpmnfiqp gafkeirlhk ltlrnnfdsl 241nvmktciqgl aglevhrlvl gefrnegnle kfdksalegl cnltieefrl ayldyylddi 301idlfncltnv ssfslvsvti ervkdfsynf gwqhlelvnc kfgqfptlkl kslkrltfts 361nkggnafsev dlpslefldl srnglsfkgc csqsdfgtts lkyldlsfng vitmssnflg 421leqlehldfq hsnlkqmsef svflslrnli yldishthtr vafngifngl sslevlkmag 481nsfqenflpd iftelrnltf ldlsqcqleq lsptafnsls slqvlnmshn nffsldtfpy 541kclnslqvld yslnhimtsk kqelqhfpss laflnltqnd factcehqsf lqwikdqrql 601lvevermeca tpsdkqgmpv lslnitcqmn ktiigvsvls vlvvsvvavl vykfyfhlml 661lagcikygrg eniydafviy ssqdedwvrn elvknleegv ppfqlclhyr dfipgvaiaa 721niihegfhks rkvivvvsqh fiqsrwcife yeiaqtwqfl ssragiifiv lqkvektllr 781qqvelyrlls rntyleweds vlgrhifwrr lrkalldgks wnpegtvgtg cnwqeatsi

In some embodiments, the biological material to be transplanted is oneor more cells or cell types, one or more tissues or tissue types, or anorgan or portion thereof. For example, the biological material to betransplanted is allogeneic biological material.

In some embodiments, the biological material to be transplanted is isletcells. In some embodiments, the islet cells are allogeneic islet cells.

In some embodiments, the biological material to be transplanted is or isderived from kidney, pancreas, liver, or intestine. For example, in someembodiments, the biological material to be transplanted is or is derivedfrom one or more hepatocytes.

In some embodiments, the anti-TLR4 antibody that is used to contact thebiological material prior to transplantation is the same anti-TLR4antibody that is administered to the subject during and/or after thebiological material has been transplanted.

In some embodiments, the anti-TLR4 antibody that is used to contact thebiological material prior to transplantation is a different antibodythan the anti-TLR4 antibody that is administered to the subject duringand/or after the biological material has been transplanted.

In some embodiments, the antibody or immunologically active fragmentthereof that specifically binds TLR4 is administered during and/or aftertransplantation in combination with one or more additional agents. Insome embodiments, the anti-TLR4 antibody and the additional agent(s) areadministered simultaneously. For example, the anti-TLR4 antibody and theadditional agent(s) can be formulated in a single composition oradministered as two or more separate compositions. In some embodiments,the anti-TLR4 antibody and the additional agent(s) are administeredsequentially.

In some embodiments, the additional agent(s) is an immunosuppressiveagent. For example, the additional agent(s) is selected frommethotrexate, cyclosporin A, tacrolimus, sirolimus, everolimus, acorticosteroid, anti-thymocyte globulin, Infliximab, Etanercept andAdalimumab. The additional agent(s) can also include any compound orother molecule that exhibits an immunosuppressive effect.

In some embodiments, the antibody is an antibody or an immunologicallyactive fragment thereof. In some embodiments, the antibody orimmunologically active fragment thereof that binds TLR4 is a monoclonalantibody. In some embodiments, the antibody or immunologically activefragment thereof that binds TLR4 is a mouse, chimeric, humanized, fullyhuman monoclonal antibody, domain antibody, single chain, F_(ab),F_(ab′) and F_((ab′)2) fragments, scFvs, or an F_(ab) expressionlibrary. In some embodiments, the anti-TLR4 antibodies also bind thehuman TLR4/MD-2 receptor complex.

In some embodiments, the antibody or immunologically active fragmentthereof that binds TLR4 comprises a variable heavy chain complementaritydetermining region 1 (V_(H) CDR1) comprising an amino acid sequence atleast 90%, 92%, 95%, 96%, 97% 98%, 99% or more identical to the aminoacid sequence of GGYSWH (SEQ ID NO: 1); a V_(H) CDR2 region comprisingan amino acid sequence at least 90%, 92%, 95%, 96%, 97% 98%, 99% or moreidentical to the amino acid sequence of YIHYSGYTDFNPSLKT (SEQ ID NO: 2);and a V_(H) CDR3 region comprising an amino acid sequence at least 90%,92%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequenceof KDPSDAFPY (SEQ ID NO: 3); a variable light chain complementaritydetermining region 1 (V_(L) CDR1) region comprising an amino acidsequence at least 90%, 92%, 95%, 96%, 97% 98%, 99% or more identical tothe amino acid sequence of RASQSISDHLH (SEQ ID NO: 4); a V_(L) CDR2region comprising an amino acid sequence at least 90%, 92%, 95%, 96%,97% 98%, 99% or more identical to the amino acid sequence of YASHAIS(SEQ ID NO: 5); and a V_(L) CDR3 region comprising an amino acidsequence at least 90%, 92%, 95%, 96%, 97% 98%, 99% or more identical tothe amino acid sequence of QQGHSFPLT (SEQ ID NO: 6). In someembodiments, the antibody or immunologically active fragment thereofthat binds TLR4 further comprises an amino acid sequence at least 90%,92%, 95%, 96%, 97% 98%, 99% or more identical to the heavy chainvariable amino acid sequenceQVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSDAFPYWGQGTLVTVSS (SEQ ID NO:7) and an amino acid sequence at least 90%, 92%, 95%, 96%, 97% 98%, 99%or more identical to the light chain variable amino acid sequenceEIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGGGTKVEIK (SEQ ID NO: 8). In someembodiments, the antibody or immunologically active fragment thereofthat binds TLR4 further comprises an amino acid sequence at least 90%,92%, 95%, 96%, 97% 98%, 99% or more identical to the heavy chain aminoacid sequence MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSDAFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9)and an amino acid sequence at least 90%, 92%, 95%, 96%, 97% 98%, 99% ormore identical to the light chain amino acid sequenceMEWSWVFLFFLSVTTGVHSEIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10).

In some embodiments, anti-TLR4 antibody or immunologically activefragment thereof is or is derived from an antibody as described inPCT/IB2005/004206, filed Jun. 14, 2005 and published as WO 2007/110678,the contents of which are hereby incorporated by reference in theirentirety.

In some embodiments, anti-TLR4 antibody or immunologically activefragment thereof is or is derived from an antibody as described in PCTapplication PCT/IB2008/003978, filed May 14, 2008 and published as WO2009/101479, the contents of which are hereby incorporated by referencein their entirety.

In some embodiments, anti-TLR4 antibody or immunologically activefragment thereof is or is derived from the anti-TLR4 antibody known asHTA125, which is described, for example, in Shimazu, et al., J. Exp.Med., vol. 189:1777-1782 (1999); Nijhuis et al., Clin. Diag. Lab.Immunol., vol. 10(4): 558-63 (2003); and Pivarcsi et al., Intl.Immunopharm., vol. 15(6):721-730 (2003), the contents of each of whichare hereby incorporated by reference in their entirety.

In some embodiments, the anti-TLR4 antibody or immunologically activefragment thereof is or is derived from a domain antibody such as, forexample, the domain antibodies that bind TLR4 described in PCTapplication PCT/EP2009/055926, filed May 15, 2009 and published as WO2009/13848, the contents of which are hereby incorporated by referencein their entirety.

In some embodiments, the anti-TLR4 antibody or immunologically activefragment thereof binds to an epitope comprising one or more amino acidresidues on human TLR4 between residues 289 and 375 of SEQ ID NO: 11.For example, in some embodiments, the antibody or immunologically activefragment thereof binds to an epitope that comprises at least residues328 and 329 of SEQ ID NO: 11. For example, in some embodiments, theantibody or immunologically active fragment thereof binds to an epitopethat comprises at least residues 349 through 351 of SEQ ID NO: 11. Forexample, in some embodiments, the antibody or immunologically activefragment thereof binds to an epitope that comprises at least residues369 through 371 of SEQ ID NO: 11. For example, in some embodiments, theantibody or immunologically active fragment thereof binds to an epitopethat comprises at least residues 328, 329, 349 through 351 and 369through 371 of SEQ ID NO: 11. For example, in some embodiments, theantibody or immunologically active fragment thereof binds to an epitopethat comprises at least residues 293 through 295 of SEQ ID NO: 11. Forexample, in some embodiments, the antibody or immunologically activefragment thereof binds to an epitope that comprises at least residues296 and 297 of SEQ ID NO: 11. For example, in some embodiments, theantibody or immunologically active fragment thereof binds to an epitopethat comprises at least residues 319 through 321 of SEQ ID NO: 11. Forexample, in some embodiments, the antibody or immunologically activefragment thereof binds to an epitope that comprises at least residues293 through 295, 296, 297 and 319 through 321 of SEQ ID NO: 11.

Pharmaceutical compositions according to the invention can include anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that depicts proliferation of mouse lymph node cellsin IMLR (Ki 67 positive cells). n=3; C=PBMC, I=islets, CI=controlisotype.

FIG. 2 is a graph that depicts mouse IFNγ secreting cells in IMLR (spotsnumber). n=3; C=PBMC, I=islets, CI=control isotype.

FIG. 3 is a graph that depicts proliferation of human PBMC in IMLR (Ki67 positive cells). n=3; C=PBMC, I=islets, CI=control isotype.

FIG. 4 is a graph that depicts human IFNγ secreting cells in IMLR (spotsnumber). n=3; C=PBMC, I=islets, CI=control isotype.

FIG. 5 is a graph that depicts blood glycaemia of transplanted mice (mMglc) where diabetic C57BL/6 mice were transplanted under the left kidneycapsule with six hundred IEQ and injected intraperitoneally twice aweek, from day 0 to day 28, with PBS (n=3), control isotype (n=6) or 5E3(n=5).

FIG. 6 is a graph that depicts graft survival in transplanted mice wherediabetic C57BL/6 mice were transplanted under the left kidney capsulewith six hundred IEQ and injected intraperitoneally twice a week, fromday 0 to day 28, with PBS (n=3), control isotype (n=6) or 5E3 (n=5).Graft rejection was defined as three consecutive blood glycaemias>18 mM.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monoclonal antibodies (mAbs) thatspecifically bind Toll like Receptor 4, and more specifically, humanTLR4. These anti-TLR4 antibodies are used in methods of inhibitingrejection of and/or prolonging survival of transplanted biologicalmaterial in a subject using antibodies that specifically bind Toll-likereceptor 4 (TLR4). Anti-TLR4 antibodies include antibodies that bind thehuman TLR4/MD-2 receptor complex and also bind TLR4 independently of thepresence of MD-2.

Exemplary antibodies of the invention include, for example, theanti-TLR4 antibodies described in PCT/IB2005/004206, filed Jun. 14, 2005and published as WO 2007/110678, the anti-TLR4 antibodies described inPCT application PCT/IB2008/003978, filed May 14, 2008 and published asWO 2009/101479, the contents of each of which are hereby incorporated byreference in their entirety, and commercially available antibodies suchas HTA125.

Exemplary antibodies of the invention include, for example, the antibodyreferred to herein as NI-0101, which is also referred to herein and inthe Figures as “hu15C1,” which binds the human TLR4/MD2 complex and alsobinds TLR4 independently of the presence of MD-2. The sequences of theNI-0101 (hu15c1) antibody are shown below, with the CDR sequencesunderlined in the VH and VL amino acid sequences:

NI-0101 Heavy Chain Nucleotide Sequence:

NI-0101 heavy chain nucleotide sequence:  (SEQ ID NO: 12)ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTACATTGCCAGGTGCAGCTTCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCACCGGTGGTTATAGCTGGCACTGGATACGGCAGCCCCCAGGGAAGGGACTGGAGTGGATGGGGTATATCCACTACAGTGGTTACACTGACTTCAACCCCTCCCTCAAGACTCGAATCACCATATCACGTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGTGGACACTGCAGTGTATTACTGTGCGAGAAAAGATCCGTCCGACGCCTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAATGCAAGGTCTCCAGTAAAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG NI-0101 heavy chain amino acid sequence:(SEQ ID NO: 9) MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSDTLSLTCAVSGYSIT GGYSWHWIRQPPGKGLEWMG YIHYSGYT DFNPSLKT RITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSDAFPY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK NI-0101 light chain nucleotide sequence: (SEQ ID NO: 13)ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACTACAGGTGTCCACTCCGAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGCAGGGTCACAGTTTTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG NI-0101 light chain amino acid sequence: (SEQ ID NO: 10)MEWSWVFLFFLSVTTGVHSEIVLTQSPDFQSVTPKEKVTITC RASQSISDHLH WYQQKPDQSPKLLIKYASHAIS GV PSRFSGSGSGTDFTLTINSLEAEDAATYYC QQGHSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The NI-0101 (hu15c1) antibody includes VH CDRs having the sequencesGGYSWH (SEQ ID NO: 1), YIHYSGYTDFNPSLKT (SEQ ID NO: 2), and KDPSDAFPY(SEQ ID NO: 3), and VL CDRs having the sequences RASQSISDHLH (SEQ ID NO:4), YASHAIS (SEQ ID NO: 5) and QQGHSFPLT (SEQ ID NO: 6).

The amino acid and nucleic acid sequences of the heavy chain variable(VH) and light chain variable (VL) regions of the anti-TLR4/MD2antibodies are shown below. The amino acids encompassing thecomplementarity determining regions (CDR) as defined by Chothia et al.1989, E. A. Kabat et al., 1991 are highlighted in underlined anditalicized text below. (See Chothia, C, et al., Nature 342:877-883(1989); Kabat, E A, et al., Sequences of Protein of immunologicalinterest, Fifth Edition, US Department of Health and Human Services, USGovernment Printing Office (1991)).

Anti-TLR4 antibodies include the antibodies described in co-pending U.S.application Ser. Nos. 11/009,939, filed Dec. 10, 2004 and 11/151,916,filed Jun. 15, 2004 and in WO 05/065015, filed Dec. 10, 2004 andPCT/US2005/020930, filed Jun. 15, 2004, each of which is herebyincorporated by reference in its entirety. Several exemplary antibodiesinclude the antibodies referred to therein as 18H10, 16G7, 15C1 and 7E3.

Anti-TLR4 antibodies include the antibodies described in co-pending U.S.application Ser. No. 11/151,916, filed Jun. 15, 2004 (U.S. PatentPublication No. US 2008-0050366 A1) and in PCT/IB2005/004206, filed Jun.15, 2004 (PCT Publication No. WO 07/110,678), each of which is herebyincorporated by reference in its entirety. The sequences of severalexemplary antibodies are shown below.

15C1 Hu V_(H) version 4-28 (SEQ ID NO: 14)

CDR 1: GGYSWH (SEQ ID NO: 1) CDR 2: YIHYSGYTDFNPSLKT (SEQ ID NO: 2)CDR 3: KDPSDGFPY (SEQ ID NO: 3) Where X₁ is Thr or SerWhere X₂ is Ile or Met Where X₃ is Val or Ile Where X₄ is Met or Ile15C1 Hu V_(H) version 3-66 (SEQ ID NO: 15)

CDR 1: GGYSWH (SEQ ID NO: 1) CDR 2: YIHYSGYTDFNPSLKT (SEQ ID NO: 2)CDR 3: KDPSDGFPY (SEQ ID NO: 3) Where X₁ is Ala or ValWhere X₂ is Val or Met Where X₃ is Leu or Phe 15C1 Hu VL version L6(SEQ ID NO: 16)

CDR1: RASQSISDHLH (SEQ ID NO: 4) CDR2: YASHAIS (SEQ ID NO: 5)CDR3: QNGHSFPLT (SEQ ID NO: 17) Where X₁ is Lys or Tyr15C1 Hu VL version A26 (SEQ ID NO: 18)

CDR1: RASQSISDHLH (SEQ ID NO: 4) CDR2: YASHAIS (SEQ ID NO: 5)CDR3: QNGHSFPLT (SEQ ID NO: 17) 18H10 Hu VH version 1-69 (SEQ ID NO: 19)

CDR1: DSYIH (SEQ ID NO: 20) CDR2: WTDPENVNSIYDPRFQG (SEQ ID NO: 21)CDR3: GYNGVYYAMDY (SEQ ID NO: 22) Where X₁ is Met or IleWhere X₂ is Lys or Thr Where X₃ is Met or Leu 18H10 Hu VL version L6(SEQ ID NO: 23)

CDR1: SASSSVIYMH (SEQ ID NO: 24) CDR2: RTYNLAS (SEQ ID NO: 25)CDR3: HQWSSFPYT (SEQ ID NO: 26) Where X₁ is Phe or Tyr7E3 Hu VH version 2-70 (SEQ ID NO: 27)

CDR1: TYNIGVG (SEQ ID NO: 28) CDR2: HIWWNDNIYYNTVLKS (SEQ ID NO: 29)CDR3: MAEGRYDAMDY (SEQ ID NO: 30) Where X₁ is Ser or ThrWhere X₂ is Ile or Phe Where X₃ is Ile or Ala 7E3 Hu VH version 3-66(SEQ ID NO: 31)

CDR1: TYNIGVG (SEQ ID NO: 28) CDR2: HIWWNDNIYYNTVLKS (SEQ ID NO: 29)CDR3: MAEGRYDAMDY (SEQ ID NO: 30) Where X₁ is Phe or AlaWhere X₂ is Val or Leu Where X₃ is Ile or Phe Where X₄ is Lys or ArgWhere X₅ is Leu or Val Where X₆ is Ile or Ala 7E3 Hu VL version L19(SEQ ID NO: 32)

CDR1: RASQDITNYLN (SEQ ID NO: 33) CDR2: YTSKLHS (SEQ ID NO: 34)CDR3: QQGNTFPWT (SEQ ID NO: 35) Where X₁ is Phe or TyrWhere X₂ is Tyr or Phe

Anti-TLR4 antibodies include the antibodies described inPCT/IB2008/003978, filed May 14, 2008 (PCT Publication No. WO2009/101479), the contents of which are hereby incorporated by referencein their entirety. These anti-TLR4 antibodies are modified to includeone or more mutations in the CDR3 portion. The sequences of severalexemplary antibodies are shown below.

15C1 humanized VH mutant 1 amino acid sequence: (SEQ ID NO: 36)QVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSDAFPYWGQGTLVTVSS15C1 humanized VH mutant 1 nucleic acid sequence: (SEQ ID NO: 37)CAGGTGCAGCTTCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCACCGGTGGTTATAGCTGGCACTGGATACGGCAGCCCCCAGGGAAGGGACTGGAGTGGATGGGGTATATCCACTACAGTGGTTACACTGACTTCAACCCCTCCCTCAAGACTCGAATCACCATATCACGTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGTGGACACTGCAGTGTATTACTGTGCGAGAAAAGATCCGTCCGACGCCTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCC15C1 humanized VH mutant 2 amino acid sequence: (SEQ ID NO: 38)QVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSEGFPYWGQGTLVTVSS15C1 humanized VH mutant 2 nucleic acid sequence: (SEQ ID NO: 39)CAGGTGCAGCTTCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCACCGGTGGTTATAGCTGGCACTGGATACGGCAGCCCCCAGGGAAGGGACTGGAGTGGATGGGGTATATCCACTACAGTGGTTACACTGACTTCAACCCCTCCCTCAAGACTCGAATCACCATATCACGTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGTGGACACTGCAGTGTATTACTGTGCGAGAAAAGATCCGTCCGAGGGATTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCC15C1 humanized VL mutant 1 amino acid sequence: (SEQ ID NO: 40)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNSHSFPLTFGGGTKVEIK15C1 humanized VL mutant 1 nucleic acid sequence: (SEQ ID NO: 41)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGAATAGTCACAGTTTTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAA A15C1 humanized VL mutant 2 amino acid sequence: (SEQ ID NO: 42)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGGGTKVEIK15C1 humanized VL mutant 2 nucleic acid sequence: (SEQ ID NO: 43)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGCAGGGTCACAGTTTTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAA A15C1 humanized VL mutant 3 amino acid sequence: (SEQ ID NO: 44)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNSSSFPLTFGGGTKVEIK15C1 humanized VL mutant 3 nucleic acid sequence: (SEQ ID NO: 45)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGAATAGTAGTAGTTTTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAA A 15C1 humanized VL mutant 4 amino acid sequence: (SEQ ID NO: 46)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSHSFPLTFGGGTKVEIK15C1 humanized VL mutant 4 nucleic acid sequence: (SEQ ID NO: 47)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGCAGAGTCACAGTTTTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAA A

DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, domain antibody, single chain, F_(ab),F_(ab′) and F_((ab′)2) fragments, scFvs, and an F_(ab) expressionlibrary.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (mAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; preferably ≦100 nM and most preferably ≦10 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to the Toll-like Receptor 4 (TLR4)/MD-2complex or to TLR4 when not complexed to MD-2, when the equilibriumbinding constant (K_(d)) is ≦1 μM, preferably ≦100 nM, more preferably≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by assayssuch as radioligand binding assays or similar assays known to thoseskilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules shown herein, andnucleic acid molecules encoding the light chain immunoglobulin moleculesshown herein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules shown herein, and the light chainimmunoglobulin molecules shown herein, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto TLR4/MD2 complex or TLR4 alone, under suitable binding conditions.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—,CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) maybe used to generate more stable peptides. In addition, constrainedpeptides comprising a consensus sequence or a substantially identicalconsensus sequence variation may be generated by methods known in theart (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example,by adding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹I, ¹²⁵I, ¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Antibodies

Monoclonal antibodies of the invention (e.g., murine monoclonal,humanized antibodies or fully human monoclonal antibodies) specificallybind TLR4. Also included in the invention are antibodies that bind tothe same epitope as the antibodies described herein. For example,antibodies of the invention that specifically bind TLR4 and/or theTLR4/MD-2 complex bind to an epitope that includes one or more aminoacid residues on human TLR4 shown below:

(SEQ ID NO: 11) 1mmsasrlagt lipamaflsc vrpeswepcv evvpnityqc melnfykipd nlpfstknld 61lsfnplrhlg sysffsfpel qvldlsrcei qtiedgayqs lshlstlilt gnpiqslalg 121afsglsslqk lvavetnlas lenfpighlk tlkelnvahn liqsfklpey fsnltnlehl 181dlssnkiqsi yctdlrvlhq mpllnlsldl slnpmnfiqp gafkeirlhk ltlrnnfdsl 241nvmktciqgl aglevhrlvl gefrnegnle kfdksalegl cnltieefrl ayldyylddi 301idlfncltnv ssfslvsvti ervkdfsynf gwqhlelvnc kfgqfptlkl kslkrltfts 361nkggnafsev dlpslefldl srnglsfkgc csqsdfgtts lkyldlsfng vitmssnflg 421leglehldfq hsnlkqmsef svflslrnli yldishthtr vafngifngl sslevlkmag 481nsfqenflpd iftelrnltf ldlsqcqleq lsptafnsls slqvlnmshn nffsldtfpy 541kclnslqvld yslnhimtsk kqelqhfpss laflnltqnd factcehqsf lqwikdqrql 601lvevermeca tpsdkqgmpv lslnitcqmn ktiigvsvls vlvvsvvavl vykfyfhlml 661lagcikygrg eniydafviy ssqdedwvrn elvknleegv ppfqlclhyr dfipgvaiaa 721niihegfhks rkvivvvsqh fiqsrwcife yeiaqtwqfl ssragiifiv lqkvektllr 781qqvelyrlls rntyleweds vlgrhifwrr lrkalldgks wnpegtvgtg cnwqeatsi

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., a murine monoclonal or humanized antibody) has the samespecificity as a monoclonal antibody used in the methods describedherein by ascertaining whether the former prevents the latter frombinding to the TLR4/MD-2 complex or to TLR4 when not complexed to MD-2.If the monoclonal antibody being tested competes with the monoclonalantibody of the invention, as shown by a decrease in binding by themonoclonal antibody of the invention, then the two monoclonal antibodiesbind to the same, or a closely related, epitope. An alternative methodfor determining whether a monoclonal antibody has the specificity ofmonoclonal antibody of the invention is to pre-incubate the monoclonalantibody of the invention with the TLR4/MD-2 complex or a soluble TLR4protein (with which it is normally reactive), and then add themonoclonal antibody being tested to determine if the monoclonal antibodybeing tested is inhibited in its ability to bind the TLR4/MD-2 complexor to bind TLR4 and TLR4 complexed with MD-2. If the monoclonal antibodybeing tested is inhibited then, in all likelihood, it has the same, orfunctionally equivalent, epitopic specificity as the monoclonal antibodyof the invention.

Use of Anti-TLR4 Antibodies

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include an anti-TLR4antibody, are used to prevent transplant rejection and/or prolongsurvival of a transplant.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating transplant rejection or othertransplant related disorders. Prolonging the survival of transplantedbiological material or otherwise preventing transplant rejection in asubject indicates that the antibody confers a clinical benefit.

Anti-TLR4 antibodies be administered in the form of pharmaceuticalcompositions. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington: The Science And Practice OfPharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co.,Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities,Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa.,1994; and Peptide And Protein Drug Delivery (Advances In ParenteralSciences, Vol. 4), 1991, M. Dekker, New York.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

In some embodiments, the antibody contains a detectable label.Antibodies are polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., F_(ab), scFv, or F_((ab)2)) isused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently-labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently-labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. Included within theusage of the term “biological sample”, therefore, is blood and afraction or component of blood including blood serum, blood plasma, orlymph. That is, the detection method of the invention can be used todetect an analyte mRNA, protein, or genomic DNA in a biological samplein vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Pharmaceutical Compositions

The antibodies or soluble chimeric polypeptides of the invention (alsoreferred to herein as “active compounds”), and derivatives, fragments,analogs and homologs thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the antibody or soluble chimeric polypeptide and apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

The Examples and data provided herein assess the role of TLR4 inmediating an immune response to allogeneic islets. Briefly, purifiedhuman or murine (DBA1) islets were co-cultured respectively withallogeneic PBMC or lymph node cells, in the presence or absence ofanti-human or anti-mouse TLR4 mAb, or relevant isotype controls.Proliferating cells were assessed using Ki67 staining, andIFNγ-secreting cells were assessed using ELISPOT assay. DBA1 islets,cultured 24 h in vitro with the anti-mouse mAbs, were transplanted underthe kidney capsule of C57BL/6 diabetic mice, injected twice a weekintraperitoneally with the anti-mouse mAbs from day 0 to 28 aftertransplantation. Blood sugar was monitored twice a week. In vitroresults showed a decrease in proliferation of 79±2% (p<0.001) and 67±16%(p=0.05) in the human and murine mixed islet-lymphocyte cultures,respectively, as compared to controls. Similarly, a decrease of 62±9%(N=3, p<0.05) and 64±10% (N=3, p<0.05) in the numbers of IFNγ-secretingcells was observed. In vivo, treatment with the anti-mouse TLR4 mAbprolonged islet graft survival to >60 days in 80% of animals (N=5),contrasting with a graft survival of 0% at 17 days in the isotypecontrol-(N=6) and buffer-treated mice (N=3). These results demonstratethat TLR4 blockade can efficiently modulate the immunogenicity of humanor murine islets in vitro and is able to achieve indefinite islet graftsurvival in vivo.

While the studies described herein use allogeneic islets, the materials,methods, and examples are illustrative only and are not intended to belimiting.

Example 1

Materials and methods for the generation of 15C1 and 5E3 monoclonalantibodies: The hu15C1 antibody, also referred to herein as NI-0101, wasgenerated and tested as described in PCT application PCT/IB2008/003978,filed May 14, 2008 and published as WO 2009/101479, the contents ofwhich are hereby incorporated by reference in their entirety. The 5E3monoclonal antibody is a monoclonal antibody that binds mouse TLR4. (SeeDaubeuf et al., “TLR4/MD-2 Monoclonal Antibody Therapy AffordsProtection in Experimental Models of Septic Shock,” J Immunol vol.179:6107-6114 (1997).

Control antibodies: Human control isotype was purchased from SIGMA® (refnumber I5029) and FITC anti-Ki-67 Set from BD Pharmingen™ (FranklinLakes N.J.).

Human islets: Pancreases were obtained from brain-dead multi-organdonors through Swisstransplant and the French Agence de la Biomédecine.

Islets were isolated using the automated method described by Ricordiwith local modifications. (See e.g., Ricordi et al., “Automated methodfor isolation of human pancreatic islets,” Diabetes, vol. 37(4): 413-20(1988); Ricordi et al., “Automated islet isolation from human pancreas,”Diabetes, vol. 38 Suppl 1:140-2 (1989)). Collagenase NB1 (ServaElectrophoresis, Heidelberg, Germany) was used. Islets were purified ona continuous Biocoll gradient (Biochrom, Berlin, Germany) with arefrigerated COBE cell processor (COBE 2991; Cobe, Lakewood, Colo.).

Islets were incubated overnight in non-adherent 60-mm diameter Petridishes containing 5 ml of CMRL-10% FCS (10% foetal calf serum, 11.2 mMglucose, 110 μg/ml sodium pyruvate and supplemented with 110 units/mlpenicillin, 110 μg/ml streptomycin, and 50 μg/ml gentamycin) with orwithout 1.5 nM 15C1 antibody or control isotype.

Animals: Two-month-old male C57BL/6 and DBA1 mice were purchased fromJanvier (Le Genest-St-Isle, France). All animals were kept in the animalfacilities at the University of Geneva with free access to food andwater. All experiments were conducted under protocols reviewed andapproved by institutional animal care and use committee.

Mouse islets: Islets of Langerhans were isolated by collagenasedigestion of pancreases from male DBA1 mice, followed by Ficollpurification using a modification of the method of Sutton et al. (Suttonet al., “Human pancreatic islet isolation with increased incubationtemperatures and variable density gradients,” Transplant Proc., vol. 22:758-59 (1990).

Islets were incubated overnight in non-adherent 60-mm diameter Petridishes containing 5 ml of RPMI 1640 complete medium (10% foetal calfserum, 11.2 mM glucose, 110 μg/ml sodium pyruvate and supplemented with110 units/ml penicillin, 110 μg/ml streptomycin, and 50 μg/mlgentamycin) with or without 1.5 nM 5E3 antibody or control isotype.

Cells extraction and Mixed Lymphocytes Cultures (MLC): Human PeripheralBlood Mononuclear Cells (PBMC) were obtained by blood centrifugation for20 min at 2000 rpm on Histopaque-1077 (SIGMA®), from healthy donorsunder written consent. Cells were immediately seeded with islets (seeMLC section).

Mouse mesenteric lymph nodes were harvested from male C57BL/6 mice andcells were extracted by manual node structural destruction. Cells wereimmediately seeded with islets (see MLC section).

Aliquots of 25 IEQ were seeded with 500′000 human PBMC or mouse lymphnode cells, in Millipore® Multiscreen-IP 96 wells plate, pre-coated withcapture IFNγ antibody (according to manufacturer recommendations,eBiosciences® human and mouse IFNγ ELISPOT Ready-SET-Go assay kits), ina total volume of 200 μl of modified complete RPMI 1640 medium (10%foetal calf or human serum, 11.2 mM glucose, 110 μg/ml sodium pyruvateand supplemented with 110 units/ml penicillin, 110 μg/ml streptomycin,0.5 mM (3-mercaptoethanol and MEM non essential amino acid solution 1×(SIGMA®)), with or without anti-TLR4 antibodies or control isotypes. Asa control, cells were seeded without islets.

After three days, cells and islets were transferred to non-adherent 96wells plates. ELISPOT membranes were revealed according to manufacturerrecommendations. Spots were counted using an automated Immunospotanalyzer (Cellular Technology Ltd, Bonn, Germany).

Reaction between cells and islets was continued during four additionaldays before cell proliferation measurement. Cells were separated fromislets by centrifugation for 1 min at 1000 rpm, fixed and permeabilizedusing eBioscience® Foxp3 Staining Buffer Set according to manufacturerrecommendations. Following 30 minutes of incubation, cells were labelledwith FITC anti-Ki-67 antibody. Cells were acquired and analyzed with aFACSCalibur flow cytometer (BDBiosciences®).

Islet transplantation: Streptozotocin (200 mg/kg i.p.) was used toinduce diabetes in C57BL/6 mice at least 5 days before islettransplantation. Diabetes was defined as nonfasting blood glucose levels≧18.0 mM for two or more consecutive days before transplantation. Afterovernight culture, 600 DBA1 mouse islet equivalents were transplantedunder the left kidney capsule of diabetic mice. Blood sugar wasmonitored twice a week and mice were injected twice a weekintraperitoneally with 500 μg of 5E3, control isotype or buffer (PBS),from day 0 to 28 after transplantation. Graft rejection was defined asthree consecutive blood glycaemias upper than 18 mM.

Presentation of data and statistical analysis: Data are presented asmean±S.E. for “n” independent experiments, and levels of significancefor differences between groups were assessed by Student's t test forunpaired groups or Log-rank (Mantel-Cox) test using GraphPad PRISMsoftware (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001).

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method of prolonging survival of transplantedbiological material in a subject, the method comprising: (i) contactingthe biological material to be transplanted with an antibody orimmunologically active fragment thereof that specifically binds aToll-like receptor 4 (TLR4) polypeptide to produce a transplantablecomposition; (ii) implanting the transplantable composition at a desiredlocation in the subject; and (iii) administering to the subject one ormore additional doses of an antibody or immunologically active fragmentthereof that specifically binds TLR4, wherein the antibody isadministered in an amount sufficient to prolong survival of thetransplanted biological material in the subject, wherein the antibody orimmunologically active fragment thereof that binds TLR4 in step (i), theantibody or immunologically active fragment thereof that binds TLR4 instep (iii) or the antibody or immunologically active fragment thereofthat binds TLR4 in step (i) and in step (iii) comprises the heavy chainamino acid sequence MGWSWIFLFLLSGTAGVHCQVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKDPSDAFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSSKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) and the light chain amino acidsequence MEWSWVFLFFLSVTTGVHSEIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10). 2.The method of claim 1, wherein the subject is a human.
 3. The method ofclaim 1, wherein the biological material to be transplanted is one ormore cells or cell types, one or more tissues or tissue types, or anorgan or portion thereof.
 4. The method of claim 1, wherein thebiological material to be transplanted is allogeneic biologicalmaterial.
 5. The method of claim 1, wherein the biological material tobe transplanted is islet cells.
 6. The method of claim 5, wherein theislet cells are allogeneic islet cells.
 7. The method of claim 1,wherein the biological material to be transplanted is or is derived fromkidney, pancreas, liver, or intestine.
 8. The method of claim 1, wherethe antibody or immunologically active fragment thereof thatspecifically binds TLR4 in steps (i) and (iii) are the same antibody orimmunologically active fragment.
 9. The method of claim 1, where theantibody or immunologically active fragment thereof that specificallybinds TLR4 in step (i) and the antibody or immunologically activefragment thereof that specifically binds TLR4 in step (iii) aredifferent antibodies or immunologically active fragments.
 10. The methodof claim 1, wherein the antibody or immunologically active fragmentthereof that binds TLR4 is a full-length fully human monoclonalantibody, a F_(ab) fragment, a F_(ab′) fragment or a F_((ab′)2)fragment, or an scFv fragment.
 11. The method of claim 1, wherein theantibody or immunologically active fragment thereof that specificallybinds TLR4 is administered in step (iii) in combination with one or moreadditional agents.
 12. The method of claim 11, wherein the one or moreadditional agents is one or more immunosuppressive agents.
 13. Themethod of claim 11, wherein the one or more additional agents isselected from methotrexate, cyclosporin A, tacrolimus, sirolimus,everolimus, a corticosteroid, anti-thymocyte globulin, Infliximab,Etanercept and Adalimumab.